UC Berkeley

Biomolecular binding interactions underpin life sciences tools that are essential to fields as diverse as molecular biology and clinical chemistry. Merging needs in life science research entail fast, robust and quantitative binding reaction characterization, such as antibody selection, gene regulation screening and drug screening. Identification, characterization, and optimization of these diverse molecular binding reactions demands the availability of powerful, quantitative analytical tools.

Among modern analysis tools well-suited to such characterizations are the techniques of electrophoresis. Electrophoretic separations physically separate molecules based on electrophoretic mobility differences among species, with mobility differences functions of molecular size, charge, and conformation. All three characteristics can depend on binding state.

Electrophoretic mobility shift assays (EMSAs) are one type of electrophoretic separations that detect binding-induced mobility changes of target analytes. In EMSAs, a probing molecule reacts with a target analyte and the binding interaction induces a change in the physicochemical properties of the target that then results in a detectable mobility shift.

EMSAs benefit from microfluidic adaption. The use of high separating electric field and miniaturized formats greatly enhance assay throughput and reduce sample consumption. Further, the precision of microfluidic control of transport and reaction confers a level of quantitation and reproducibility that are difficult (if not impossible) to achieve with conventional tools.

Our group has previously introduced a microfluidic EMSA (μMSA) assay that reduces reagent consumption ten-fold and processing time a hundred-fold. While a notable advance, microchip based EMSAs suffer from equipment-heavy infrastructure needs and serial electrophoresis implementations, limiting throughput and scale-up potential.

To surmount these limitations of microfluidic EMSAs, our group has pioneered “open-microfluidic” electrophoresis arrays that support >384 concurrent polyacrylamide gel electrophoresis (PAGE) separations. The PAGE molecular sieving gels are photo-patterned directly on a planar substrate – not inside of enclosed microfluidic channels. The adaption of EMSAs to such a PAGE gel array format reduces infrastructure demands and affords parallel operation, thereby overcoming the shortcomings of in-channel glass devices.

Here we report on the design, development, characterization, optimization, and application of free-standing polyacrylamide gel (fsPAG) EMSAs to answer questions about molecular binding fundamental to molecular biology research and the biotechnology industry. We harness the open, multiplexed nature of the fsPAG format, the quantitative precision of fine fluidic control and the small sample volume requirements to yield two sets of analytical contributions.

The first set of contributions centers on discerning both form and function during RNA riboswitch binding to metabolites. Not only does the RNA riboswitch bind to certain metabolites, the molecule takes on a compact conformation if that binding event is functional. This compact conformation results in an electrophoretic mobility shift versus the non-function RNA riboswitch. We first developed a microchip based rapid in-vitro cyclic-di-GMP biosensor. This assay builds on the previously reported riboswitch μMSA technology and enables fast (30 min) cyclic-di-GMP concentration determination in cell extracts with high detection sensitivity. Our work is the only “minimalist cyclic-di-GMP biosensor” reported so far, which performs direct concentration measurements with no need for complex riboswitch derivative construction.

We then characterized fsPAG EMSAs for riboswitch binding analysis. We detailed the fundamental physical properties of the open microfluidic gel array and utilized the analytical tool for HTP riboswitch binding analysis. fsPAG EMSAs offer a throughput (10 data/min) that is 30 times higher than our own previously reported μMSA and 1000 higher than the canonical slab-gel EMSAs.

In a second set of contributions, we applied the precision quantitation capability of fsPAG EMSAs to report binding kinetics of fragment antigen-binding antibody reagents. We integrated the open-microfluidic fsPAG with an acoustic sample delivery system and developed a novel automated binding affinity measurement tool for fragment antigen-binding fragment (Fab) molecules. To date, the assay offers the highest reported throughput. Important to such throughput and to reproducibility, the assay eliminates the cumbersome manual sample loading previously involved in performing fsPAGE and greatly improves the electrophoretic uniformity of the assay. The equilibrium constants of 6 Fab were simultaneously measured on a 384-plex fsPAG device.

In a more speculative and forward-looking contribution, we designed and prototyped an fsPAG western blot assay; a departure from in-channel design strategies our laboratory has pursued in the past. A critical contribution of the protype assay is sample stacking during transfer from the PAGE separation to the blotting step; with the stacking enhancing the detection sensitivity and reduceing the assay time. The fsPAG western blot benefits from using the molecular binding interactions we have characterized earlier, but now in open-microfluidic format.

Taken together, we have designed, developed, and applied high-throughput molecular binding analysis platforms with open-microfluidic polyacrylamide gel electrophoresis tools to both detection of functional riboswitch binding events and quantitative characterization of antibody fragment binding kinetics. Fundamental and design findings offer new understanding and capabilities in parallelized binding reaction analyses and affinity based molecular screening, fulfilling two sets of unmet needs in bioanalytical technology.